Einführung in die Genetik - Technische Universität München€¦ · Einführung in die Genetik -...

Einführung in die Genetik

Prof. Dr. Kay Schneitz (EBio Pflanzen)http://[email protected]

Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/[email protected]

“Downloads” is linked to Schneitz web page

Friday, December 9, 11

http://plantdev.bio.wzw.tum.de




mailto:[email protected]






Einführung in die Genetik - InhalteEinführung in die Genetik - InhalteEinführung in die Genetik - Inhalte1 Einführung 18. 10. 11 KS2 Struktur von Genen und Chromosomen 25. 10. 11 KS3 Genfunktion 08. 11. 11 KS4 Transmission der DNA während der Zellteilung 15. 11. 11 KS5 Vererbung von Einzelgenveränderungen 22. 11. 11 KS6 Genetische Rekombination (Eukaryonten) 29. 11. 11 KS7 Genetische Rekombination (Bakterien/Viren) 06. 12. 11 KS8 Rekombinante DNA-Technologie 13. 12. 11 CS9 Kartierung/Charakterisierung ganzer Genome 20. 12. 11 CS

10 Genmutationen: Ursache und Reparatur 10. 01. 12 CS11 Veränderungen der Chromosomen 17. 01. 12 CS12 Genetische Analyse biologischer Prozesse 24. 01. 12 CS13 Transposons bei Eukaryonten 31. 01. 12 CS14 Genexpression und Onkogene 07. 02. 12 CS


Genetic Recombination in Bacteria and their Viruses

Genetics 07


Summary• Plasmids

• small DNA circles (1-2% of bacterial DNA), replicate autonomously in bacterial cell

• contain additional genes (e.g, resistance genes, F genes)

• Conjugation

• directional transfer of DNA from a donor to a recipient cell, requires physical contact

• F plasmid confers “maleness”

• Hfr strains

• copy of F plasmid integrated somewhere in bacterial chromosome

• produces high number of recombinants in Hfr x F- crosses

• merozygote exconjugants where multiple crossovers can occur between exo- and endogenote

• Interrupted mating and recombination mapping

• circular genetic map of E. coliFriday, December 9, 11

Summary• Bacteriophages

• bacterial viruses

• Virulent phages

• immediately lyse and kill their host bacterium

• e.g., bacteriophages P1, T4

• Temperate phage

• maintained in host bacterium without immediately killing the host

• e.g., bacteriophage λ

• Prophage

• phage genome that is integrated into the host chromosome

• lysogenic bacterium carries a prophage

• General transduction

• Phage transfers any piece of bacterial genomic DNA between cells

• Special transduction

• Prophage integrated at a single, specific site in bacterial chromsome (e.g., λ attachment site)

• transfers only genes located close by the attachment siteFriday, December 9, 11

Recombinant DNA Technology

Genetics 08


Molecular cloning

DNA sequencing 1

Examples

DNA sequencing II

Polymerase chain reaction

Recombination-based cloning


Molecular cloning - what for?

• to sequence them (individual genes or whole genomes)

• to put them in an order (genomes and genome fragments)

• to do something with them (express their gene products/proteins)

• to manipulate them (make gene fusions, introduce mutations)

• ...


Cloning for DNA sequencing

• Mutations in the BREAST CANCER1 (BRCA1) gene

Sequencing requires the clonal amplification of the DNA fragments to be sequenced


Cloning for genome assembly

• Sorted fragments of an assembled genome

Sequencing requires the clonal amplification of the DNA fragments to be sequenced


Cloning for genetic engineering

• Protein expression in a heterologous host (bacteria)

Recombinant protein expression requires cloningFriday, December 9, 11

Cloning for cell biology

• Protein localization in a (plant) cell using green fluorescent protein

Gene fusions can be generated by molecular cloningFriday, December 9, 11

Molecular cloning


Cloning and cloning vectors

Restriction digest

Ligation

Transformation

Clonal amplification

DNA preparation

Selection

Restriction digestor sequencing


Restriction digest

Ligation

Transformation


DNA preparation

Selection


Restriction sites

Origin of replication (ori)

Antibiotic resistance(amp, tet)



restriction sites(polylinker, multiple cloning site)

(lac promoter)

origin of replication (ori)

selection(for plasmid)

(lacZ reporter)

selection(for insert)



Restriction enzymes

• there are hundreds of restriction enzymes available commercially

• restrcition enzymes can generate different types of overhangs

• there is a restriction enzyme basically for every DNA sequence

• the frequency depends on the length of the recognition site (e.g. for a hexameric site the frequency is on average 4096 bp)


Restriction enzymes

Digest of genomic DNAM size marker

1,3 undigested

2,4 digested

Digest of plasmid DNAM size marker

1,3 undigested

2,4 digested

Agarose gel Agarose gel


• restriction enzymes are isolated from bacteria

• bacteria have restriction enzmyes to protect themselves against foreign DNA

• they protect their own DNA by DNA methlyation

Restriction enzymes


T4 DNA Ligase



restriction sites(polylinker, multiple cloning site)

(lac promoter)

origin of replication (ori)

selection(for plasmid)

(lacZ reporter)

selection(for insert)



Restriction digest

Ligation

Transformation


DNA preparation

Selection


Restriction sites




Transformation

Plasmid after ligation

Heat shock transformation(Alternative: electro shock) Selection



Restriction digest

Ligation

Transformation


DNA preparation

Selection


Restriction sites




DNA preparation


DNA sequencing


The dideoxy sequencing method (1)

Nobel prize in 1980Walter Gilbert and Frederick Sanger(Sanger sequencing)


The dideoxy sequencing method (2)


(Fluorescent) dye terminator technology


Examples


Cloning vectors for sequencing


Cloning GST-fusion constructs

• Expression and purification of a GST-tagged protein

• GST, glutathione S-transferase (protein)

• glutathione, small molecule that can be bound to a resin

GE Healthcare

pGEX vectors, GST Gene Fusion System

imagination at work

Map of the glutathione S-transferase fusion vectors showing reading frames and main features. Even though stop codons in all three frames are not depicted in this map, all thirteen vectors have stop codons in all three frames downstream from the multiple cloning site.

Ordering information

Product Quantity Code no.pGEX-‐1λT EcoRI/BAP 5 µg 28-‐9546-‐56

pGEX-‐2T 25 µg 28-‐9546-‐53

pGEX-‐2TK 25 µg 28-‐9546-‐46

pGEX-‐3X 25 µg 28-‐9546-‐54

pGEX-‐4T-‐1 25 µg 28-‐9545-‐49

pGEX-‐4T-‐2 25 µg 28-‐9545-‐50

pGEX-‐4T-‐3 25 µg 28-‐9545-‐52

pGEX-‐5X-‐1 25 µg 28-‐9545-‐53

pGEX-‐5X-‐2 25 µg 28-‐9545-‐54

pGEX-‐5X-‐3 25 µg 28-‐9545-‐55

pGEX-‐6P-‐1 25 µg 28-‐9546-‐48

pGEX-‐6P-‐2 25 µg 28-‐9546-‐50

pGEX-‐6P-‐3 25 µg 28-‐9546-‐51

Do you want to learn more? Read the GST Gene Fusion System Handbook (18-‐1142-‐75). Please contact your local GE Healthcare representative for a printed copy.

pGEX-1!T

pGEX-6P-1

EcoRI SmaI SalI XhoI NotIBamHI

PreScission™ Protease

Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro HisCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT

pGEX-6P-2

BamHI

PreScission Protease

Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG

EcoRI SmaI SalI XhoI NotI

pGEX-6P-3

BamHI

PreScission Protease

Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly ArgCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC

EcoRI SmaI SalI XhoI NotI

BamHI EcoRI SmaI SalI XhoI NotI






EcoRI

CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA

BamHI

Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp

Thrombin

Stop codons

pGEX~4900 bp

pBR322ori

BalI

BspMI

Ptac

calI q

NarI

EcoRV

BssHII

BstEII MluI

ApaI

Tth111I AatII

PstI

p4.5AlwNI

pSj10 Bam7Stop7!

pGEX-4T-2

pGEX-5X-1

pGEX-5X-2

pGEX-5X-3

pGEX-4T-1

pGEX-4T-3

pGEX-3X

pGEX-2TK

Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

Stop codon

Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

Stop codons

Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

Stop codon

Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

Stop codons

Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspCTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

Stop codons

Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspCTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

Stop codons

ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GACIle Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser

Stop codons

Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val

Kinase

CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGAStop codons

Thrombin

Thrombin

Thrombin

Thrombin

Factor Xa

Factor Xa

Factor Xa

Factor Xa

EcoRIBamHI SmaI

EcoRIBamHI SmaI

pGEX-2T

CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACGLeu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp

Stop codonsEcoRI

Thrombin

BamHI SmaI

glutathione S-transferase

Amp r

Acrylamide protein gel (SDS-PAGE)


Cloning GFP-fusions

• Expression of a GFP-tagged protein for cell biological studies

• GFP, green fluorescent protein

• GFP can be seen based on its fluorescent propertiesFriday, December 9, 11

DNA sequencing II


Next generation sequencing


Third generation sequencing

http://www.youtube.com/watch?v=q8gK4P7jg_sFriday, December 9, 11

http://www.youtube.com/watch?v=q8gK4P7jg_s

http://www.youtube.com/watch?v=q8gK4P7jg_s

Polymerase chain reaction (PCR)


Polymerase Chain Reaction (1)

Melting of DNA 95 °C

Primer annealing ca. 55 °C

Primer extension 72 °C


Primer extension72 °C

Melting of DNA95 °C






Melting of DNA 95 °C



...and so forth



Typical PCR Program

Step 1: Melting of DNA 95 °C - 4 min

Step 2: Melting of DNA 95 °C - 1 minStep 3: Primer annealing 55 °C - 1 minStep 4: Primer extension 72 °C - 1 min per kb

go to Step 2 - 29 times

Step 5: Final primer extension 72 °C - 5 minStep 6: Storage 4 °C forever


Primer (oligonucleotide) orders


Taq polymerase - a heat stable polymerase

Yellowstone National Park

Thermus aquaticus

Taq polymerase

Karry MullisNobel prize 1993


PCR machinesthree waterbaths - three temperatures


PCR machines - the real thing


What is so cool about PCR?

• highly sensitive amplification of desired gene fragment possible

• amplification without propagation of the cloned fragments in bacteria: Good enough for sequencing and cloning! But flanking DNA sequences have to be known for primer design!

• primers can be designed to insert restriction sites

• primers can be designed to introduce desired sequence changes (mutations)

• ...


Cloning PCR products• vectors for the cloning of blunt-ended or T-tailed PCR products

are available

• use Topoisomerase for ligation (TOPO cloning)



• vector system for the cloning of the same gene insert into different destination vectors

• use recombinases for gene integration


The end


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